1. Field of the Invention
The present invention relates generally to a rapid screening process for measuring selected gas production of photosynthetic algal clones, and relates more specifically to a method for screening individual photosynthetic algal clones for gaseous fuel production capacity by illuminating individual algal colonies grown on a solid growth medium and measuring selected gases produced therefrom.
2. Description of the Prior Art
It is well known that certain species of algae cultivated under anaerobic conditions are capable of hydrogen photoproduction. The gas producing capabilities and anaerobic metabolism of Scenedesmus unicellular algae and other species, for example, have been investigated. (See H. Gaffron and J. Rubin, "Fermentative and Photochemical Production of Hydrogen in Algae", J. Gen. Physiol., 219-240 (1942) and J. J. Brand, "Survey of Hydrogenase Activity in Algae", Final Report for Subcontract No. B-1-9229-1 under Prime Contract No. EG-77-C-01-4042, University of Texas at Austin) Different algal species, and different strains of the same species exhibit varying degrees of hydrogen photoproduction, or none at all, and past efforts to measure photoproduction of different algal species and strains have been cumbersome and time consuming. Quantification of hydrogen production is important, since algal photoproduction represents potential for gaseous fuel production from renewable inorganic resources.
One common method for screening algae to determine their hydrogen producing capabilities involves growing algae in a liquid culture medium and measuring the hydrogen evolved with a reverse-biased Clark-type oxygen electrode. This method is described in R. Wang, F. P. Healy and J. Myers, "Amperometric Measurement of Hydrogen Evolution in Chlamydomonas", Plant Physiol. 48, 108-110 (1970) and R. T. Wang, "Amperometric Hydrogen Electrode", in "Methods in Enzymology" 69, 409-413 (1980), Academic Press. According to this screening method, the algae cultured in liquid medium are grown in a culture vessel, and hydrogen and oxygen gas produced by an appropriate aliquot of the algae are confined to a static volume. As hydrogen and oxygen gas concentrations increase, algal photoproduction is inhibited and hydrogenase activity is reduced. This inhibitory effect varies with individual algal strains, and therefore cannot be quantified generally. Calibration of the reverse-biased Clark electrode is difficult and measurements of hydrogen produced are often inaccurate due to the electrochemical instability of the reverse-biased Clark electrode.
U.S. Pat. No. 4,442,211 teaches a method for measuring hydrogen production of algae cultured in an aqueous phase. Algae grown in a liquid culture medium are sequentially subjected to anaerobic conditions, aerobic conditions, and then anaerobic conditions again to provide an enhanced rate of hydrogen and oxygen gas production. Hydrogen and oxygen gas production are measured by illuminating the cell culture in an anaerobic environment, passing an inert gas over the culture as a carrier for removing the hydrogen and oxygen gases produced, and then determining product gas composition by means of hydrogen and oxygen gas sensors. One drawback of this screening method is that the entire volume of the liquid culture is irradiated to measure the collective gas production of all cells in the culture and the gas production of individual cells and strains within the culture volume cannot be determined. For example, mutant cells may have developed with greatly enhanced hydrogen producing capabilities, but the presence of these cells is not detectable since the gaseous production of the bulk culture volume is measured. This screening method is also limited from the standpoint that pure algal cultures must be maintained to determine the gas production capacity of different strains and species, and pure cell cultures are difficult to isolate and maintain.
U.S. Pat. No. 4,010,076 teaches hydrogen production by stabilized photometabolically active microbes grown on a support medium such as agar gel wherein an aqueous substrate solution flows through the reactor under anaerobic conditions in the presence of light and hydrogen gas produced is passed to a collection vessel. According to the teachings of this patent, microorganisms are immobilized on a solid support to facilitate separation of the gaseous product from the microorganisms.
Hydrogen photoproduction of algal cultures is dependent upon cellular hydrogenase activity, and measurement of hydrogenase levels in algal strains and species would, therefore, give some indication of hydrogen production capacity. Enzymatic assays, however, typically require destruction of the living cells and are very time consuming. A colony screening method for isolation of hydrogenase regulatory bacterial mutants wherein bacterial colonies are grown on a solid agar medium and transferred to filter paper discs for screening is taught in H. G. Schlegel and M. Meyer, "Isolation of Hydrogenase Regulatory Mutants of Hydrogen-Oxidizing Bacteria by a Colony-Screening Method", Arch. Microbiol. 141, 377-383 (1985). Detection of hydrogenases in bacterial colonies transferred to filter paper discs is determined by soaking the discs in a dye solution and exposing them to hydrogen gas. Dye reduction indicates the presence of hydrogenase containing colonies.